Research The Immune Protective Effect Of Chicken Gallinac Recombine Lactobacillus Preparation

Defensin has the characteristics of wide antimicrobial spectrum, non-toxic side effects for people and animals, low immunogenicity, no drug resistance, no residual, good solubility and thermal stability, not sensitive to high ionic strength and pH change, so the defensin has the potential alternatives to traditional antibiotics.β-defensin, an antimicrobial peptide in animals, shows not only antimicrobial and antiviral activity but also the function of enhance immunity, a higher research and development use potential, It is one of the focus for many scholars.In this study with chicken gallinac(Gal-6) as the research object, according to the sequence of Gal-6 in the genebank to synthesize Gal-6,build plasmid pUC57-T-Gal-6 what contains Gal-6. Double restriction enzymes digestion for the previous and shuttle expression vector. Connection to build pSIP409-Gal-6 plasmid, into e.coli DH5α to identified, the correct recombinant plasmid electroporated into Lactobacillus plantarum NC8. Constructing recombinant Gal-6 lactobacillus, after analysising by SDS-PAGE, Western blotting to detect Gal-6 expression, Application of thin layer agar diffusion method to determine the bacteriostatic activity of recombinant lactobacillus. The results showed successful Constructing recombinant plasmid pSIP409-Gal–6 and electroporated into Lactobacillus plantarum, express success. Bacteriostatic activity experiments prove that Gal- 6 respectively on staphylococcus aureus and escherichia coli has certain antibacterial activity.To further analyze the immune protective effect of recombinant lacticacid bacteria, we verify whether Gal-6 can enhance the chick immune function, enhance resistance to disease and infection. Recombinant lactobacillus pSIP409-Gal-6 was feeded chicks after SppIP induced.The 45 2-3-week-old chicks were randomly divided into three groups, 15 each group, the three groups were pSIP409-Gal-6 group, pSIP409 group and PBS group. Recombinant lacticacid bacteria 5.0×109 CFU oral immune chicks, immune dose is 0.3 mL every chick,immune three weeks, the fourth week stop to immune,the fifth week enhance the immune. After the immune cycle, chicks were stoped to immunity, and were blooded in the wings to detect CD4+and CD4+T lymphocytes by flow cytometry. Two weeks after the interval, pathogenic E. coli ETEC 5.0×109 CFU attack was using for attacking drug experiments. Then we monitor body weight and change of weight in each group per day.The chicks were killed and collected duodenum, ileum and cecum to produce pathological sections for observing the intestinal pathological changes after bacterial attack 10 days. The results showed that weight loss of the pSIP409-Gal-6 group and pSIP409 group were less than the PBS group.From the results of flow cytometry, we can know that Gal-6 can increase the number of CD4+ lymphocytes and enhance immunity. From the results of intestinal pathological sections after inoculation,we can know that oral recombinant lactobacillus pSIP409-Gal-6 can improve the intestinal pathological changes. β-defensin to express, through lacticacid bacteria in animals, enhance the animals’ ability to resist infection, there is significance to the animal food safety and public health,it plays a positive role for reduce and replace the use of antibiotics.